The amplification and detection of specific nucleic acid sequences present in minute amounts is an increasingly important technique for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, studying genetic susceptibility to disease, and measuring response to various types of treatment. Such procedures have also found expanding uses in detecting and quantitating microorganisms in foodstuffs, environmental samples, seed stocks, and other types of material where the presence of specific microorganisms may need to be monitored. Other applications are found in the forensic sciences, anthropology, archaeology, biology and clinical medicine where measurement of the relatedness of nucleic acid sequences has been used to identify criminal suspects, resolve paternity disputes, construct genealogical and phylogenetic trees, aid in classifying a variety of life forms, and identify disease states.
A common method for detecting and quantitating specific nucleic acid sequences is nucleic acid hybridization. The sensitivity of nucleic acid hybridization assays is limited primarily by the specific activity of the probe, the rate and extent of the hybridization of the probe, and the sensitivity with which the label can be detected. The most sensitive procedures may lack many of the features required for routine clinical and environmental testing, such as speed, economy and convenience. Furthermore, their sensitivities may not be sufficient for many desired applications.
As a result of the interactions among the various components and component steps of this type of assay, there is often an inverse relationship between sensitivity and specificity. Thus, steps taken to increase the sensitivity of the assay (such as increasing the specific activity of the probe) may result in a higher percentage of false positive test results. The linkage between sensitivity and specificity has been a significant barrier to improving the sensitivity of hybridization assays. One solution to this problem would be to specifically increase the amount of target sequence present using an amplification procedure. Amplification of a unique portion of the target sequence without amplification of a significant portion of the information encoded in the remaining sequences of the sample could give an increase in sensitivity while at the same time not compromising specificity.
Amplification has been used to increase the sensitivity of nucleic acid assays. One common method for specifically amplifying nucleic acid sequences termed the "polymerase chain reaction" or "PCR" has been described by Mullis et al. (See U.S. Pat. Nos. 4,683,202 and 4,683,195 and European patent applications 86302298.4, 86302298.4, and 87300203.4 and Methods in Enzymology, Volume 155, 1987, pp. 335-350.) The procedure uses repeated cycles of primer dependent nucleic acid synthesis occurring simultaneously using each strand of a complementary sequence as a template. Therefore, at least two primers are required in PCR. The sequence amplified is defined by the primer molecules that initiate synthesis. The primers are complementary to the 3' -end portion of a target sequence or its complement and must complex with those sites in order for nucleic acid synthesis to begin. After extension product synthesis, the strands are separated, generally by thermal denaturation, before the next synthesis step. In the PCR procedure, copies of both strands of a complementary sequence are synthesized.
The requirement of repeated cycling of reaction temperature between several different and extreme temperatures is a disadvantage of the PCR procedure.
The PCR procedure has been coupled to RNA transcription by incorporating a promoter sequence into one of the primers used in the PCR reaction and then, after amplification by the PCR procedure for several cycles, using the double-stranded DNA as template for the transcription of single-stranded RNA. (see e.g., Murakawa et al. DNA 7:827-295 (1988)). Other methods of amplifying nucleic acid sequences are also commercially available. These methods include the ligation amplification reaction (LCR), and the transcription-based amplification reaction. Ligation amplification reaction is described by Wu, D. Y and Wallace, R. B, Genomics 4:560-569 (1989) and Barringer, K. J., et al., Gene 89:117-122 (1990). Transcription-based amplification reaction is described by Kwoh, D. Y., et al., Proc. Natl. Acad. Sci. USA 86:1173-1177 (1989). These methods have the advantages of high sensitivity, but the disadvantages of being prone to false-positive results from reaction product contamination.
It is therefore an object of the present invention to amplify a target nucleic acid by continuous amplification reaction, which does not require repeated cycles of amplification and produces many RNA copies of the target sequence.
Another object of the present invention relates to detection of minute amounts of nucleic acids through use of a continuous amplification reaction (also referred to herein as "CAR").
Yet another object of the invention is to indirectly amplify a target DNA signal by synthesizing and detecting multiple copy RNA molecules.
It is a further object of the present invention to provide a cost-effective, sensitive, solution hybridization assay for RNA transcripts produced by CAR.